PROJECT SUMMARY/ABSTRACT Spinal cord injury (SCI) debilitates many patients every year and leaves them with few effective therapies to improve their conditions. Researchers consider interdisciplinary therapeutic approaches, combining the application of biomaterials, growth-promoting proteins, anti-inhibitory molecules to reduce the growth-inhibitory environment, and cell transplantation to form new connections for motor recovery. A promising population, interneurons (INs) have been found to rewire the spinal cord after injury in rodent models, improving functional recovery. Our lab has focused on generating these cell populations of the ventral spinal cord, including motor neurons (MNs). Lab members have developed induction protocols to guide mouse embryonic stem cells (mESCs) to form a few of these populations: MNs, V2a INs, and V3 INs. The goal of the research proposed in this study is to determine the therapeutic potential of V0 INs in a rat model of SCI. To complete this work, two aims are described. Aim 1 seeks to enable purification of V0 INs from mESC induction cultures and is expected to require a year to complete. Transgenic mESC lines will be generated through CRISPR/Cas9-mediated recombination of selection cassettes into a gene marker for V0 progenitors. Selection cassettes will include one containing an antibiotic resistance enzyme to allow for antibiotic-mediated purification and another containing a fluorescent reporter to allow for lineage tracing of the desired population. Selection conditions will be tested to obtain the highest purity V0 IN population, and cells will be characterized by transcript and protein expression and electrophysiological properties to verify their similarity to endogenous V0 INs. Using a combinatorial approach, V0 INs will be analyzed for their potential to recover motor function in a rat SCI model for Aim 2, which is expected to be completed in a year and a half. High purity V0 INs will be transplanted in a hyaluronic acid based matrix containing growth factors to support their survival and growth. Rats will undergo behavioral testing to observe the extent of functional recovery. Spinal cord tissue analysis will be used to determine the growth and penetration of the transplanted cells at the site of injury. It is expected that Aim 1 will produce two selectable transgenic mESC lines that can be induced and purified for V0 INs similar to endogenous V0 INs. For the rat SCI model of Aim 2, it is expected that rats receiving a combination of matrix, growth factors, and transplanted high purity V0 INs will show the greatest improvement in motor recovery over other groups tested (with/without cells and/or growth factors). If successful, these studies will lend insight into cell therapies to improve motor outcomes after SCI.